Cysteine desulfurases perform essential functions in releasing sulfur from cysteine, forming a covalent persulfide, and channeling the persulfide sulfur to biologically critical recipients e.g. ironsulfur (Fe-S) clusters and thiolated tRNAs. The activity of cysteine desulfurase cannot be replaced by exogenously supplied sulfide or by other sulfur metabolizing enzymes, and thus cysteine desulfurase is essential for cell viability. The eukaryotic cysteine desulfurase is encoded by a single nuclear gene and is found primarily in mitochondria. In Saccharomyces cerevisiae mitochondria, the cysteine desulfurase protein Nfs1 is assembled in a large protein complex with a recently identified special accessory protein called Isd11. We found that Isd11 is required for Nfs1 cysteine desulfurase activity. Thus, Nfs1 and Isd11 expressed together in bacteria are assembled into an active complex, while these components expressed separately are inactive. Here we propose to characterize the active Nfs1/Isd11 complex, and regulation of its cysteine desulfurase activity by Fe-S cluster scaffold proteins, Isu. Aim 1 is to determine the role of yeast Isd11 in the active Nfs1/Isd11 cysteine desulfurase complex. Experiments will be performed using bacterial expressed and purified proteins, and also in a more physiological context using isolated intact mitochondria. Aim 2 is to characterize formation of persulfide sulfur on Nfs1/Isd11 in isolated intact mitochondria, with focus on the regulatory effect of the D37A mutation of Isu. The significance derives from the essential role of cysteine desulfurase for viability of all human cells. The enzyme is found primarily in mitochondria and is required for Fe-S cluster synthesis and tRNA thiolation in mitochondria, both are essential processes. The identification of specific diseases arising from defective Fe-S cluster assembly (e.g. Friedreich's ataxia, sideroblastic anemia, and mitochondrial myopathy) represents the tip of the iceberg. The recent discovery of a key role of Fe-S clusters in DNA repair and genome stability suggests that cysteine desulfurases are involved in these processes.